Proper development and function of the eye, and indeed of an entire living organism, involves the correct and precise expression of a very large number of genes located on the organism's chromosomes. A great amount can be learned about these genes and their effects through in vitro laboratory experimentation and tissue culture techniques, however their role and importance in the development and health of an intact organism can be assessed only when studied in an intact organism. A myriad of knowledge has been obtained from transgenic mice, gene knockout and knockin mice. We have previously generated alpha-crystallin gene knockout mice to study the in vivo function of these remarkable proteins. The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. Lenticular alpha-crystallin is comprised of two similar subunits alphaA and alphaB, each encoded by a single gene. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperone activity, autokinase activity, and interact with, and affect the state of, several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. Toward understanding the major roles of alpha-crystallin in vivo, we previously generated alphaA- and alphaB-crystallin gene knockout mice and alphaA-/alphaB-crystallin gene double knockout mice (DKO). The lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus. We showed that morphological abnormalities in the lens secondary fiber cells of DKO mice are consistent with, and likely result from, elevated DEVDase and VEIDase activities, corresponding to caspase 3 and caspase 6 respectively. Activity levels of caspase 3 and caspase 6 in DKO mouse lenses fluctuated with the animal?s age, and changes in caspase activities were consistent with changes in lens morphology. TUNEL staining revealed differences between DKO and WT lenses. TUNEL-positive nuclei in WT lenses were present in a narrow band in the lens cortex in the area of organelle loss. In DKO mice at 7 weeks of age, almost every nucleated lens fiber cell, regardless of its position in the lens, was TUNEL-positive. Moreover, the signal intensity was greater than that seen in WT, suggesting a higher level of DNA fragmentation. Regions of morphological change, or cell loss, in lenses from DKO mice coincide with intense TUNEL signal, suggesting an apoptotic character of cell disintegration. Based on our screening experiments with a wide spectrum of caspase inhibitors, caspase 6 plays a similar, or even more important, role than caspase 3 in secondary lens fiber cells maturation, consistent with recent literature reports. However, modulation of caspase 6 by alpha-crystallin had not been previously described. Employing a pull-down assay we were able to demonstrate direct interaction between caspase 6 and alphaA-crystallin. Interestingly, using same assay, we did not find any interaction between caspase 6 and alphaB-crystallin. These data are consistent with our observation that lenses of alphaA-, but not alphaB-crystallin, single KO mice display secondary lens fiber cell disintegration, but only in much older animals. Alpha-crystallin had not previously been implicated in regulation of this executioner caspase. Perhaps alphaA-crystallin controls activity of caspase 6, which appears to be more important for lens fiber cell maturation, and alphaB-crystallin controls activity of caspase 3. Although interactions between alphaA-crystallin and caspase 3 have not yet been reported, we cannot rule out modulation of caspase 3 activity by alphaA-crystallin. Our data suggest that alpha-crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity following degradation of mitochondria and other organelles, which occurs during the apoptosis-like pathway of lens cell terminal differentiation. The mechanism by which alphaA- and alphaB-crystallin inhibit caspase activity is unknown. Data suggest possible pathways of inhibition: alphaB-crystallin could interact with factors promoting apoptosis, e.g. Bax and Bcl-Xs, or the interaction could be directly between caspase 6 and alphaA-crystallin and between caspase 3 and alphaB-crystallin. Further elucidation of the mechanisms regulating caspase activities is essential. A project initiated last year to do a large (50 kb) targeted deletion in the mouse genome, spanning 5 closely related genes expressed in the visual system, failed to produce a correctly targeted ES cell clone in the 300 clones tested. In collaboration with the laboratories of Xuejun Wang and Nikola Golenhofen we are further investigating the role of alphaB-crystallin and HSPB2 in cardiac function. In collaboration with Allen Taylor, we are examining the role of the ubiquitin pathway in lens development. In collaboration with Ram Kannan and colleagues, we are examining the role of alpha-crystallin in the retina and RPE.